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- Table of Contents
CHRM3 / Muscarinic acetylcholine receptor M3 · Western blot design guide
Design a Western Blot for CHRM3
Source-linked CHRM3 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CHRM3 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled September 2026 · For research use; verify linked source records and product datasheet before use
CHRM3 Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~66.1 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Skin (IHC candidate; verify WB) +4 more | |
| Negative control | Appendix (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked CHRM3 Western Blot Protocol Options
The M03150 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human fetal brain lysate (catalog M03150) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M03150; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected CHRM3 Western Blot Band Size?
CHRM3 has a predicted monomer size of 66.1 kDa; glycosylation and homodimerization could affect migration, but no observed band or demonstrated shift is supplied.
| Band near 66.1 kDa | Consistent with the predicted CHRM3 monomer if identity is confirmed |
| Band above 66.1 kDa | Could reflect N-linked glycosylation, though its effect on migration is unmeasured |
| Band near twice the monomer size | Could reflect a CHRM3 homodimer retained during sample preparation |
| Broad band or smear | Could reflect heterogeneous N-linked glycosylation if confirmed experimentally |
💡Expected CHRM3 appearanceUniProt predicts a 66.1 kDa CHRM3 monomer; glycosylation and homodimerization could affect migration, but no empirical band size is supplied, so confirm band identity with appropriate controls.
| Predicted CHRM3 mass | 66.1 kDa is the calculated monomer size, not measured migration |
| N-linked glycosylation at Asn5 | May increase apparent mass; the effect is unmeasured |
| N-linked glycosylation at Asn6 | May increase apparent mass; the effect is unmeasured |
| N-linked glycosylation at Asn15 | May increase apparent mass; the effect is unmeasured |
| N-linked glycosylation at Asn41 | May increase apparent mass; the effect is unmeasured |
| N-linked glycosylation at Asn48 | May increase apparent mass; the effect is unmeasured |
| CHRM3 homodimer formation | May produce a band near twice the monomer size if the dimer persists during sample preparation |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated CHRM3 may be poorly extracted | Check membrane solubilization and compare with a membrane-enriched fraction |
| Band higher than expected | N-linked glycosylation or a retained homodimer may affect migration | Compare glycosidase treatment and sample preparation conditions; verify band identity |
| Broad smear instead of sharp band | Possible heterogeneous N-linked glycosylation | Compare untreated and glycosidase-treated samples |
| Multiple bands | Possible glycosylation differences or retained CHRM3 oligomers | Compare glycosidase treatment and sample preparation conditions; verify band identity |
| Weak or no signal | Membrane extraction may yield little CHRM3 | Check extraction and include a positive CHRM3 sample |
Experiment evidence
Western blot images showing CHRM3 band
M03150 · CHRM3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CHRM3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CHRM3 in Western blot, you can use skin tissue, where HPA reports high expression.
Positive control: Skin (IHC candidate; verify WB)
Negative control: Appendix (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: CHRM3 is a multi-pass membrane protein, so membrane-enriched lysate may improve detection.
HPA tissue expression evidence for CHRM3
Comprehensive Human Protein Atlas IHC scoring per tissue. Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Higher expression tissues · candidate positive controls from IHC
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Skin | fibroblasts | High | Protein (IHC) | HPA → |
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
| Caudate | neuronal cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | Medium | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cervix | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CHRM3 Western Blot Tips
Deeper troubleshooting and optimisation questions for CHRM3, answered from its protein features.
How should CHRM3 band migration be interpreted?
Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.
Is a second CHRM3 band evidence of another isoform?
Isoforms · The supplied features list one isoform and no alternative sequence. Do not assign a second band to an annotated CHRM3 isoform on this evidence alone.
Which CHRM3 glycosylation sites matter when assessing band patterns?
PTM · UniProt lists N-linked glycosylation at Asn 5, 6, 15, 41, and 48. These are UniProt sequence coordinates; check the numbering convention before comparing them with an antibody or paper. Their presence does not predict how many bands will appear.
UniProt annotates phosphoserine at position 385 in its sequence numbering. A band change alone cannot establish phosphorylation at this site; compare samples with an appropriate site-specific or phosphatase-based check if testing that explanation.
UniProt annotates phosphoserine at position 385 in its sequence numbering. A band change alone cannot establish phosphorylation at this site; compare samples with an appropriate site-specific or phosphatase-based check if testing that explanation.
Does this guide establish induction of CHRM3?
Induction · No general induction response is established by this guide. A pathway or gene-set association is not evidence of induction in a particular specimen. Verify the relevant treatment and control in a target-specific experiment.
How should transfer be checked for CHRM3?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M03150 datasheet where specified. Otherwise compare 5% milk or 5% BSA in TBST; for a phospho-specific assay start with BSA. Optimize background and specific signal with matched controls.
How should CHRM3 bands be quantified?
Quantitation · Define the band or band region consistently across samples and report which signal was measured. CHRM3 has annotated glycosylation, phosphorylation, and oligomerization, so record distinct bands separately until their identities are established. The supplied evidence does not identify an empirical reference band.
Should CHRM3 migrate at its predicted 66.1 kDa?
Interpretation · 66.1 kDa is the predicted mass, not an observed Western blot position. CHRM3 has five annotated N-linked glycosylation sites, but these features alone do not establish a visible shift or explain any difference from 66.1 kDa. No empirical band position was supplied.
CHRM3 is annotated as a homodimer, and dimers can form tetramers. Consider these forms when interpreting higher bands, but the annotation does not show that oligomers persist under your Western blot conditions.
Two disulfide bonds are annotated. If band patterns differ between reducing and nonreducing preparations, consider disulfide-dependent structure as one explanation. The supplied features do not predict a specific band position or shift.
CHRM3 is annotated as a homodimer, and dimers can form tetramers. Consider these forms when interpreting higher bands, but the annotation does not show that oligomers persist under your Western blot conditions.
Two disulfide bonds are annotated. If band patterns differ between reducing and nonreducing preparations, consider disulfide-dependent structure as one explanation. The supplied features do not predict a specific band position or shift.
Boster reagents
CHRM3 Western Blot Antibodies
Catalog antibodies with Western blot application and product-specific WB images. Evaluate suitability with the reported sample, controls and experimental conditions.
Real WB data
Anti-Muscarinic Acetylcholine Receptor M3 Rabbit Monoclonal Antibody
Cat # M03150
The catalog reports one anti-CHRM3 rabbit monoclonal antibody, M03150, with reported human reactivity. Its Western blot image shows M3 expression in human fetal brain lysate. The supplied evidence does not establish performance in other tissues or species.
Which to pick: M03150 is the only listed option. Its Western blot image uses human fetal brain lysate, making that the documented sample context to compare with your experiment.
Source: BosterBio CHRM3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P20309.
- Human Protein Atlas. CHRM3 tissue expression.
- PMC12289679 — target-verified WB comparison
- PMC10783849 — target-verified WB comparison
- PMC7831931 — target-verified WB comparison